Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence

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Reference

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence

NYONDA, Mary, et al.

Abstract

Toxoplasma gondii infects virtually any nucleated cell and resides inside a non‐phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of Immunity Regulated GTPases (IRGs) and Guanylate Binding Proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine‐threonine kinases ROP17 and ROP18, and a pseudo‐kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM.

Here, we characterize GRA60, a novel dense granule effector which localizes to the vacuolar space and PVM and contributes to virulence of RH in mice suggesting a contribution to the subversion of host cell defense mechanisms. Members of the host cell IRG defense system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs [...]

NYONDA, Mary, et al. Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence. Cellular Microbiology, 2020, p. e13278

PMID : 33040458

DOI : 10.1111/cmi.13278

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence

Mary Akinyi Nyonda¹, Pierre-Mehdi Hammoudi¹, Shu Ye¹, Jessica Maire¹, Jean-Baptiste Marq¹, Masahiro Yamamoto² and Dominique Soldati-Favre^1*

Affiliation

1Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland

2Department of Immunoparasitology, Division of Infectious Diseases, Osaka University, Japan

*Correspondance: Dominique Soldati-Favre (dominique.soldati-favre@unige.ch)

Keywords: Apicomplexa, Toxoplasma gondii, parasite, interferon gamma, dense granule,

effector, virulence, host-parasite interaction, Immunity related GTPases.

Running title: T. gondii GRA60 interferes with host Irgb10 and Irga6 action

Summary

Toxoplasma gondii infects virtually any nucleated cell and resides inside a non-phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of Immunity Regulated GTPases (IRGs) and Guanylate Binding Proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine-threonine kinases ROP17 and ROP18, and a pseudo-kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM.

Here, we characterize GRA60, a novel dense granule effector which localizes to the vacuolar space and PVM and contributes to virulence of RH in mice suggesting a contribution to the subversion of host cell defense mechanisms. Members of the host cell IRG defense system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RH∆rop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RH∆rop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and is found to associate with ROP18, a member of the virulence complex.

Introduction

Successful perpetuation of the life cycle of a pathogen depends on its capacity to effectively transmit to a new host, and its ability to escape onslaught by host immune responses.

Pathogens tend to counteract host defenses by mounting and executing immune evasion or modulation tactics. A dynamic host and pathogen co-evolution process ensues as a result of selection pressures imposed on both parties. Host immunity is a driving force behind pathogen evolution and a determining factor of pathogen diversity but is also continually reshaped by the nature of antigens presented. The phylum of Apicomplexa groups human and animal pathogens, including Toxoplasma gondii, a parasite prevalent in one-third of the world’s population, causing toxoplasmosis (Jones et al., 2012). This parasite has a complex yet flexible lifecycle comprising three different developmental stages. Although T. gondii can infect various warm-blooded animals as intermediate hosts, predation of mice infected with cysts containing bradyzoites by felids, the definitive host, is central to completion of the lifecycle and transmission. Proceeding an active invasion process of a host cell, T. gondii secludes itself within a non-fusogenic but communicative parasitophorous vacuole (PV) enclosed by a PV membrane (PVM) within which it safely divides until it is ready to egress and infect new host cells (Clough et al., 2017). The infective tachyzoite efficiently discharge ROPs and GRAs effector proteins from two secretory organelles the rhoptries and dense granules, respectively.

The GRAs reside in the PV lumen, at the external surface of the PVM and are also targeted to the host cytosol or nucleus where they interact with host proteins modulating host signaling pathways and transcription levels (Hakimi et al., 2017).

Recent studies have advanced our knowledge on how immunity is elicited against T. gondii

tachyzoites, focusing on the mouse model, a natural intermediate host. In mice, innate defenses are mediated by Toll like receptors (TLRs) activation, specifically TLR11 and TLR12 which can detect tachyzoite profilin-like protein (Koblansky et al., 2013; Yarovinsky et al., 2005). This stimulates interleukin-12 (IL-12) production by TLR-activated dendritic cells and inflammatory monocytes, an important step that shapes adaptive immunity. In turn, IL-12 drives the production of IFN-γ by natural killer cells and T-lymphocytes. IFN-γ subsequently induces the expression of numerous genes including the GTPases family which encodes effector proteins central to host cell autonomous defenses (Hunter et al., 2012; Sasai et al., 2019). One class of these effector proteins is the Immunity Related GTPases (IRGs) or p47 GTPases. They are markedly expanded in rodents, encoded by 23 genes compared to only 2 genes in humans. The IRGs play a crucial role of T. gondii elimination and hence contribute to host survival (Bekpen et al., 2005; Howard et al., 2011).

The IRGs possess a universally conserved canonical nucleotide binding motif GX4GKSand are classified as GKS proteins which include IRGA, IRGB, IRGC and IRGD subclasses.

However, a few have a GX4GMS motif in place of the canonical sequence and are named GMS proteins. The GMS members are the endomembrane localized Irgm1, Irgm2 and Irgm3 proteins that function as negative regulators of the GKS effectors (Bekpen et al., 2005; Haldar et al., 2013; Hunn et al., 2008). A proportion of T. gondii PVMs rapidly become coated with the Irgb6, Irgb10, Irga6, Irgm2, Irgm3 and Irgd proteins shortly post invasion (Khaminets et al., 2010), progressing to vesiculation and eventual rupture of the PVM (Y. O. Zhao et al., 2009).

Subsequently, tachyzoites are eliminated and the infected cell dies via necrosis (Martens et al., 2005). A second class of IFN-γ induced effector proteins that can bind to and vesiculate T.

gondii PVM are the Guanylate Binding Proteins (GBPs) or p65 GTPases (Kravets et al., 2016).

Humans encode 6 GBPs while mice possess 11 GBPs. Human GBP1 (hGBP1) and mouse

GBP2 (mGBP2) are paralogs that become isoprenylated allowing their translocation to intracellular membranes and may have similar mechanisms of action (Nantais et al., 1996).

During invasion and in readiness to counter host immune onslaught, the parasites secrete a myriad of ROPs and GRAs used to orchestrate subversion of the host immune response via signaling or directly at the PVM-host cytosol interface. Specifically, the pseudo kinase ROP5 (Behnke et al., 2015; Etheridge et al., 2014; Fleckenstein et al., 2012; Niedelman et al., 2012;

Reese et al., 2014), the serine-threonine kinases ROP17 and ROP18 (Behnke et al., 2015;

Etheridge et al., 2014; Fentress et al., 2010; Fleckenstein et al., 2012; Hermanns et al., 2016;

Niedelman et al., 2012; Steinfeldt et al., 2010) as well as the dense granule protein GRA7, (Alaganan et al., 2014) work in concert to bind to and phosphorylate an array of IRGs. Once phosphorylated, IRGs are unable to hydrolyze GTP and cannot form oligomers, hence IRG loading onto the PVM is disabled (Steinfeldt et al., 2010). GRA12, on the other hand, resides within the PV and contributes to both parasite virulence and establishment of chronic infection through modulation of IFN-γ mediated immune responses without involving the GTPase effectors (Fox et al., 2019; Wang et al., 2020).

To date, these are the only T. gondii virulence factors described to directly target the IRGs and allow parasite escape of the host innate immune mechanisms (Hunter et al., 2012). These effectors have also proven to be active in a strain specific manner. ROP18 contributes to the difference in virulence between type I, II, and III lineages as it is down-regulated in the avirulent type III strain (Saeij et al., 2006; Taylor et al., 2006). In addition, ROP5/II allele is different from those found in type I/III strains which are similar and can functionally complement each other (Reese et al., 2011). GRA15 alongside ROP16 and ROP18 play a role by affecting mGBP1 recruitment at the PVM (Selleck et al., 2013; Virreira Winter et al., 2011), whilst ROP54 inhibits mGBP2 recruitment all in type I strain (Kim et al., 2016). Remarkably, under IFN-γ regulated

conditions GRA15/II interacts with TRAF2 and TRAF6 ubiquitin ligases in human foreskin fibroblasts and mouse embryonic fibroblast (Mukhopadhyay et al., 2020). It promotes lysosomal destruction in human cells and enhances loading of Irgb6, GBPs, Ubiquitin and LC3B to boost parasite clearance in mouse cells (Mukhopadhyay et al., 2020).

The breadth and complexity of the mouse IRG and GBP resistance systems plausibly impose an evolutionary pressure on T. gondii that in counterpart evolves virulence factors and mechanisms to neutralize the host cell autonomous defense system. Dense granule proteins group functionally diverse class of effectors that frequently exhibit intrinsically disordered regions (IDRs), characterized by a low degree of conservation prone to accommodate mutations. Enrichment of IDRs on effector proteins increase structural flexibility, presumably facilitating their translocation across cellular barriers, such as the PVM. This phenomenon has equally been described for secretion of effector proteins in several pathogenic bacteria (Hakimi et al., 2017; Marin et al., 2013).

In this study, we applied genome mining to identify effector proteins and identified a novel dense granule protein GRA60 that carries 3 IDRs and is secreted to the PV space and PVM.

GRA60 is implicated in the neutralization of Irgb10 and Irga6 loading but not Irgb6 nor mGBP2 at the PVM of IFN-γ activated MEFs. Consequently, type I RH parasites lacking GRA60 (RH∆gra60) show an increased clearance compared to wildtype parasites in murine but not in human cells. In mice, RH∆gra60 parasites exhibit a reduced virulence with a significant prolonged time before death compared to RH. This significant alteration of virulence is not observed in type II lineage ME49∆gra60 and establishment of chronic infection is only mildly reduced. These findings attest for GRA60 being a key component of the parasite repertoire of virulence factors contributing to the high virulence observed in the type I strain.

Results

TGME49_204270 encodes a dense granule protein referred to as GRA60

Global mapping of T. gondii proteome using the spatial proteomics method termed hyperplexed Localization of Organelle Proteins by Isotopic Tagging (hyperLOPIT), predicts the existence of at least 171 GRAs and 129 ROPs with a majority still having unknown functions (ToxoLOPIT) (Barylyuk et al., 2020). Genome mining in ToxoDB for IDRs in polymorphic proteins anticipated to be secreted led to identification of 20 hypothetical genes including TGME49_204270 herein named GRA60 (Figure S1 and Table S1). Of the 20, HyperLOPIT predictions assigned two proteins to the dense granules, one to the rhoptries, five to the cytosol while the remaining twelve were unassigned (Table S1). The annotation of TGME49_204270 predicts a hypothetical protein of 544 amino acids, bearing a signal peptide, three IDRs and a transmembrane domain (Figure 1a). GRA60 is also present and conserved in the closely related cyst-forming coccidian parasites Neospora and Hammondia. There are no homologues in other genus of the Apicomplexa phylum or other organisms (Figure S2a).

The first step taken to characterize GRA60 was to add 3-Ty epitope tags at the C-terminus of the protein by homologous recombination in the endogenous locus (Figure S2b), followed by confirmation of integration by genomic PCR (Figure S2c). Indirect immunofluorescence assay on extracellular parasites revealed that GRA60-Ty colocalized with GRA1, a marker of dense granules (Figure 1b). IFAs on intracellular parasites stained to detect GAP45, a parasite periphery marker distinctly localized GRA60-Ty inside the parasite and within the vacuole.

GRA60-Ty showed a dual localization to the PV space and the PVM, which was confirmed by

partial colocalization with GRA3 (Figure 1b). The association of GRA60 with the PVM was examined by fractionating whole cell extracts of human foreskin fibroblasts (HFFs) co-infected with GRA60-Ty and CDC50.1mAID.3HA strains (Bisio et al., 2019), followed by western blot analysis. As expected, the integral membrane protein CDC50.1 remains tightly bound to membranes in the insoluble fraction in presence of sodium carbonate solution but becomes fully soluble in 1% Triton X-100 (Bisio et al., 2019). GRA60-Ty is detectable as a major band below 70 kDa, corresponding to the predicted molecular weight (Figure 1c) and is found to be partially extracted by sodium carbonate, as previously reported for the PVM associated GRA17 (Gold et al., 2015). GRA60-Ty is partially and fully extracted in presence of 1% Triton X-100 and RIPA buffer, respectively (Figure 1c). In extracellular parasites, GRA60-Ty exists in two pools, half soluble and insoluble in PBS. Nevertheless, we observed similar membrane association properties as intracellular parasites when examined in sodium carbonate, 1%

Triton X-100 and RIPA buffer solutions. These data suggest that GRA60-Ty is partially soluble, and membrane bound. We subsequently investigated the topology of GRA60-Ty at the PVM.

We selectively permeabilized the host plasma membrane but not the PVM using 0.001%

saponin detergent. GAP45 antibodies were used to assess whether the PVM was permeabilized. We consistently observed that GAP45 negative vacuoles were also negative for Ty antibody staining (Figure 1d), indicating that the C-terminus of GRA60 is facing the PV lumen. We could only detect the GRA60-Ty when the vacuole was permeabilized (Figure 1e).

To confirm this observation, we used ROP2,3,4 antibodies. ROP2 associates with the PVM and is exposed to the host cytosol (Beckers et al., 1994; Rome et al., 2008). We could not detect GRA60-Ty on ROP2,3,4 positive non-permeabilized vacuoles (Figure 1d and e). These findings demonstrate that GRA60 is inserted in the PVM with its C-terminus facing the PV lumen and its N-terminus, the host cytosol (Figure 1f). Similar topology has been described for

type 1 transmembrane GRA5 (Lecordier et al., 1999).

GRA60 is dispensable for parasite development in human cells but is important for growth in murine cells

To learn the function of GRA60, mutant strains lacking the GRA60 gene were generated in both type I and type II (ME49) parental lines using the CRISPR-Cas9 genome editing strategy (Figure S3a and S3b) (Shen et al., 2014). RH∆gra60 was complemented with the endogenous promoter and coding sequence of GRA60 at the locus to generate the RH∆gra60/GRA60- Ty.HXGPRT.DHFR strain (RH∆gra60/GRA60-Ty) (Figure S3c, S3d and S3e). Deletion of GRA60 had no impact on the lytic cycle progression in type I and II strains in vitro, illustrated by comparable sized plaques formed 7 and 12 days post infection on confluent HFFs in comparison to the respective parental strains (Figure 2a). ∆gra60 parasites in both type I and II strains showed normal intracellular replication rates at 24 hours and 40 hours post infection (Figure 2b). To rule out possible minor impact on parasite fitness, competition assays were carried out by mixing with wild type GFP-expressing parasites as an internal control. ∆gra60 type I and II parasites displayed normal progression in comparison to the respective parental strains (Figure 2c).

T. gondii tachyzoites show marked synchronicity during cellular division within a vacuole that occurs via endodyogeny leading to two daughter cells emerging from a single mother cell (Francia et al., 2014). The synchronized division is conferred by the connection between intravacuolar parasites that is dependent on myosin I and J (Frenal et al., 2017). We used antibodies against IMC1, a protein that localizes in both mature and daughter cells’ inner membrane complex (IMC). IFA on infected HFFs revealed that 50% of ∆gra60 parasites were

growing asynchronously (Figure 2d and 2e), suggesting that the communication between parasites might be altered. Interestingly, RH∆gra60 parasites grew slower in both IFN-γ activated and non-activated Mouse Embryonic Fibroblasts (MEFs). We observed a significant increase in number of vacuoles with one parasite and fewer with eight parasites in RH∆gra60 compared to RH with or without IFN-γ stimulation (Figure 2f and Table S3). This marked difference in growth pattern between the two cell types could possibly be attributed to the diversity in IFN-γ mediated responses between MEFs and HFFs.

Deletion of GRA60 attenuates parasite virulence during acute infection and reduces cyst burden during chronic infection

The contribution of GRA60 to parasite virulence in vivo was examined by intraperitoneal injection in C57BL/6 strain of mice with either type I or II ∆gra60 tachyzoites. Remarkably, while mice infected with 200 RH tachyzoites took a median of 9 days to succumb, RH∆gra60 exhibited a median of 17 days (Figure 3a). This difference in survival curve pattern was statistically significant (p= 0.0255) (Table S3). In parallel, we infected a similar number of mice with a lethal dose of 1,000 ME49 tachyzoites. Mice infected with ME49 succumbed while 40%

of those injected with ME49∆gra60 survived (Figure 3b), however this difference in survival was not statistically significant (p= 0.1266) (Table S3). The number of cysts per brain of chronically infected mice was determined 4 weeks post intraperitoneal injection with a sub- lethal dose of 250 ME49∆gra60 or ME49 tachyzoites (n=8, per group). A notable reduction in cysts burden was observed in the sacrificed mice, ME49∆gra60 infected arm (Median, 650 cysts) compared to ME49 infected group (Median, 1533) (Figure 3c). Equal viability of test strains used to infect mice was established by plaque assay (Figure 3Sf). In order to address

the concern that the reduction in cyst numbers observed in vivo was due to attenuated virulence, CRISPR/CAS9 strategy was applied to replace the GRA60 promoter region with the promoter sequence of the tachyzoite abundantly expressed and bradyzoite repressed gene SAG1 in the cyst prone ME49 strain, (Di Cristina et al., 2017; Di Cristina et al., 2008; Krishnan et al., 2020; Shen et al., 2014) to produce ME49pSAG1/GRA60 strain (Figure S4 a-b).

Subsequently, 3-Ty epitopes were added to the C-terminus of GRA60 of both ME49 and ME49pSAG1/GRA60 strains to generate ME49GRA60-Ty and ME49pSAG1/GRA60-Ty parasite lines (Figure S4 b). Clones were confirmed by PCR followed by gel electrophoresis and IFAs using mouse anti Ty antibodies (Figure S4 c and d). IFAs on ME49pSAG1/GRA60- Ty showed that SAG1 promoter sequence insertion did not alter the PV and PVM localization of GRA60 at tachyzoites stage (Figure S4 d). IFA using the cyst wall marker Dolichos biflorus lectin (DBA) on 7 days in vitro differentiated ME49pSAG1/GRA60-Ty bradyzoites (Weiss et al., 1995) showed a cyst periphery and matrix staining of GRA60 (Figure S4 e). Mander’s correlation coefficient of proportion of GRA60 that overlap with DBA for ME49pSAG1/GRA60- Ty was (M1=1.0) (Table S3). Comparative quantification from western blot of GRA60 between ME49pSAG1/GRA60-Ty and ME49GRA60-Ty in both stages revealed GRA60 protein amounts to be significantly increased in ME49pSAG1/GRA60-Ty tachyzoites and decreased significantly upon conversion to bradyzoites (Figure S4 f-h, Table S3). Noticeably, GRA60 in ME49pSAG1/GRA60-Ty remained unexpectedly higher than endogenous levels (Figure S4 f- h, Table S3). ME49pSAG1/GRA60 parasites grew normally compared to wild type in vitro illustrated by equally sized plaques fixed 12 days post infection (Figure S4 i). GRA60 contribution to chronicity was assessed in mice (n=8 per group) by infection with equally viable ME49 and ME49pSAG1/GRA60 strains (Figure S4 j ). Survival of animals infected with ME49pSAG1/GRA60 was 75% versus 50% of mice infected with ME49 strain (Figure S4 k).

The surviving mice were euthanized 4 weeks post infection and the tissue cysts enumerated.

We observed a significant reduction in cyst burden (Median, 850 cysts) in the ME49pSAG1/GRA60 group, compared to the ME49 infected group (Median, 3636) (Figure 3d, Table S3). Two of the ME49 infected mice with the highest cyst counts were euthanized before the end of experiment due to apparent severe symptoms. The cyst burden in vivo was comparable to amounts obtained from mice infected with ME49∆gra60 despite indistinct in vitro downregulation of GRA60 by SAG1 promoter. Taken together, these noteworthy findings implicate GRA60 as a crucial virulence factor in T. gondii type I and II strains, important for both acute and chronic infection stages.

Mouse Irgb10 and Irga6 but not Irgb6 are recruited at the vacuole of RH∆gra60 in IFN-γ stimulated cells

The attenuation of virulence in RH∆gra60 during acute infection is comparable to that of RH∆gra7 (CD1 mice) and RH∆rop18 (C57BL/6 mice) (Alaganan et al., 2014; Selleck et al., 2013). Reasonably, given the established position of GRA7 and ROP18 in neutralizing the host IRG defense system, we comprehensively examined the potential role of GRA60 in blocking IRG recruitment at the PVM and its possible interaction with ROP5 and ROP18. GRA60 gene was deleted in both RH∆rop18 and RH∆rop5 parasites by use of CRISPR-Cas9 genome editing, to generate RH∆rop18∆gra60 and RH∆rop5∆gra60 strains respectively (Figure S5a and S5b) (Shen et al., 2014). The individual and double knockout parasites grew normally as observed from the plaque sizes of each mutant compared to the parental line (Figure S5c), consistent with a previous study that had shown that RH∆gra7 and RH∆rop18 grew normally in HFFs (Alaganan et al., 2014). To assess the recruitment of IRGs at the PVM of these

mutants, C57BL/6 MEFs were seeded on coverslips and stimulated for 20 hours with 10000 U/ml of mouse IFN-γ prior to infection. ME49, RH∆rop18 and RH∆rop5 were used as positive controls and RH as a negative control for IRG recruitment at the PVM. We also used the RH∆gra60/GRA60-Ty strain to confirm gene function. Consistent with the decrease in virulence observed for RH∆gra60 parasites in mice, 32% of RH∆gra60 vacuoles became coated with the rapidly loaded Irgb10 compared to only 15% of RH vacuoles (p= 0.004) (Figure 4a-b and S6a). A notable addition, 45% of RH∆gra60 vacuole were coated with Irga6 in comparison to only 9% of RH vacuoles (p= 0.001) (Figure 4c-d and S6b). We observed reversion to low levels similar to RH in loading of Irgb10 and Irga6 using the RH∆gra60/GRA60- Ty strain showing values of 12% and 5% respectively (Figure 4b and 4d), signifying GRA60 function had been restored. The presence of these IRGs at the PVM of ME49, RH, RH∆rop5 and RH∆rop18 were as reported in the past (Figure 4a-4d and S6a-b) (Y. Zhao et al., 2009).

Interestingly, and unlike what was seen with Irga6 and Irgb10, deletion of GRA60 had no effect on the loading of Irgb6 but the positive controls showed recruitment (Figure 4e-f and S6c).

Our investigations as to whether GRA60 could act in concert with ROP5 and ROP18 revealed a modest but insignificant increase in levels of Irgb10 on parasite PVMs, from 39% in RH∆rop5 to 47% in the RH∆rop5∆gra60 double mutant. Comparably, recruitment of Irga6 at the PVM of the double-knockout mutants was not additive. 59% of RH∆rop5 were coated with Irga6 while RH∆rop5∆gra60 had 53% of its vacuoles become Irga6 positive. Similarly, 53% and 54% of vacuoles in RH∆rop18 and RH∆rop18∆gra60 respectively became loaded with Irga6. (Figure 4a-4d). From these data, we reasoned that GRA60 presumably primarily targets Irgb10 and consequently Irga6. GRA60 potentially cooperates with ROP5 in targeting Irgb10, plausibly without ROP18 involvement. To exclude that RH∆gra60 phenotype is indirect due to the mis-

localization of ROP5, ROP18 and GRA7 to the PVM, we quantified ROP5, ROP18 and GRA7 proteins detected by IFA staining at the PVM of RH∆gra60 in comparison RH parasites 10 mins post infection. We observed no significant differences in fluorescence for the three proteins in absence of GRA60 (Figure 5a-e, Table S3). IRGs start to accumulate on the PVM as early as 2.5 mins post invasion, exponentially increase between 15-30 mins and can still de detected 2 hours post invasion (Khaminets et al., 2010). Therefore, presence of GRA60 at the PVM interface would be paramount at this early time point. GRA60 secretion kinetics and presence at the PVM were subsequently investigated at 5, 10, 30 and 60 mins post parasite invasion. ROP1, a rhoptry bulb protein that is discharged during the invasion process and becomes associated with the newly developed PVM, was used as an early secreted protein marker (Saffer et al., 1992). GRA60 could be detected as early as 5 mins post invasion and partially colocalized with ROP1 at the PVM of the newly invaded parasites (Figure 5b).

Evaluations of the colocalization correlation coefficients of GRA60 overlap to ROP1 protein at the PVM were carried out using Mander’s colocalization coefficient (MCC) methods (Dunn et al., 2011). At 5 minutes, the Manders' Coefficients of the fraction of GRA60 overlapping ROP1 was M1=0.81, and M1=0.56 for the images displayed and M1=0.473 at 60 minutes post infection (Table S3). This delineates that GRA60 is secreted early upon host entry which is congruent with the timing of IRG proteins relocation to the PVM and aligns with the observed IRG recruitment in its absence. It has also been observed that other members of the virulence complex are secreted early (Etheridge et al., 2014). However, deletion of GRA60 does not interfere with the presence of ROP5, ROP18 or GRA7 at the PVM.

GRA60 interacts with ROP18

Given that in absence of GRA60, Irgb10 and Irga6 proteins become recruited to the PVM without affecting ROP5 and ROP18 localization at this frontier, it became fundamental to determine how GRA60 influences the IRG resistance pathway. To probe for possible molecular interactions, GRA60 was co-immunoprecipitated (co-IP) from RHGRA60-Ty infected cells in the presence of 1% Triton X-100 using Ty antibodies in three independent experiments. This was followed by western blot analysis using ROP18 antibodies. The efficiency of GRA60 immunoprecipitation was demonstrated by probing with Ty antibodies (Figure 6a). We illustrate a ROP18/GRA60 interaction via co-immunoprecipitation of ROP18 which migrated around 55kDa (Figure 6b) in the RHGRA60-Ty infected HFF cells (Figure 6c). The IRG-targeting complex constitutes ROP5, ROP18, ROP17 and GRA7 (Etheridge et al., 2014; Hermanns et al., 2016). Association of GRA60 with ROP18 alludes to it being an additional component of the T. gondii virulence complex.

Loss of Irga6 recruitment at the PVM in absence of Irgm1 and Irgm2 proteins

Previous work reported that deletion of the GMS proteins (Irgm1 and Irgm3) causes dysregulation and mis-localization of the GKS counterparts, predictably leading to impaired IRGs loading at the PVM of T. gondii vacuoles (Hunn et al., 2008) (Figure 7a). Next, we aimed to uncover whether GRA60 activity is dependent on specific regulation of Irga6 by Irgm1 and Irgm2. For this, we used ∆Irgm1, ∆Irgm2 and ∆Irgm1/∆Irgm2 MEFs to examine the loading of Irga6 onto RH∆gra60 vacuoles. We invariably used the ME49 strain, RH∆rop18 and RH∆rop5 as positive controls and RH as a negative one. Strikingly, a consistent and significant reduction of Irga6 loading on ME49 and RH∆gra60 vacuoles was noted. In RH∆gra60, Irga6 recruitment decreased from 45% in MEFs to 6%, 7% and 3% in ∆Irgm1, ∆Irgm2 and ∆Irgm1∆Irgm2 cell

lines respectively (Figure 7b and Table S3). Interestingly, only modest reduction in recruitment was observed with the positive controls (RH∆rop18 and RH∆rop5) in either ∆Irgm1 or ∆Irgm2 cell lines, however, the depletion became significant in ∆Irgm1/∆Irgm2. In RH, the basal recruitment observed in MEFS reduced inconsiderably in ∆Irgm1, ∆Irgm2 and ∆Irgm1∆Irgm2 cell types. (Figure 7b and Table S3). These results implicate an absolute reliance of GRA60 activity on functional Irgm1 and Irgm2 proteins.

GRA60 plays no role in the recruitment of mGBP2 at the PVM

The recruitment of mouse guanylate binding protein 1 (mGBP1) to T. gondii PVM is hindered by ROP5 and ROP18 (Selleck et al., 2013). We proceeded to probe if GRA60 could negatively affect the localization of mGBP2 at the PVM. IFN-γ activated MEFs were infected for 90 mins with test strains RH∆gra60 and RH∆gra60/GRA60-Ty. We consistently used the ME49 strain, RH∆rop18 and RH∆rop5 as positive controls and RH as a negative one and then assessed mGB2 recruitment by IFA. Strikingly, RH∆gra60 and RH scarcely exhibited recruitment of mGBP2 giving an average of 0.7% and 0.2% positive vacuoles respectively. Contrarily, the positive controls (ME49, RH∆rop18 and RH∆rop5) presented recruitment at (14.5%, 10.6%

and 18.7%) respectively (Figure 8a-b). The levels of mGBP2 loading are comparable to other studies (Degrandi et al., 2013). In consequence, we surmised that GRA60 is not implicated in preventing mGBP2 recruitment at the PVM of type I T. gondii tachyzoites.

Discussion

The presence of extended stretches of IDRs is abundant in secreted effectors in bacteria (Marin et al., 2013). Similarly, in T. gondii, the exported GRAs such as GRA16, GRA24, GRA15, GRA18, IST and TEEGR have a high propensity to possess IDRs which presumably facilitate protein translocation across the PVM (Braun et al., 2019; Gay et al., 2016; Hakimi et al., 2015; He et al., 2018; Olias et al., 2016). GRA60 has been identified as an intrinsically disordered protein, holding three N-terminal IDRs with stretches longer than 50 amino acids, a SP and a C-terminal TM. GRA60 exists both as soluble and insoluble pools in the dense granules suggesting that the hydrophobic domain remain partially unexposed as previously described for GRA2 and GRA5 (Braun et al., 2008). In infected cells, GRA60 is secreted in the PV and found partially soluble only in presence of detergent, presumably due to the anchorage in the PVM. Similar solubility profile was reported for GRA17, a PVM resident protein (Gold et al., 2015). With regard to the topology of PVM associated transmembrane GRAs, the IDRs tend to face the host cytosol (Stavropoulos et al., 2012). We observe that GRA60 is tethered to the PVM with the C-terminal portion facing the PV lumen while the larger N-terminal segment bearing the IDRs is exposed to the host cytosol. The IDRs are typically enriched in phosphorylation sites, involved in various interactions (Burgi et al., 2016; Iakoucheva et al., 2004). In this context, TgWNGI, a parasite specific kinase, has been shown to phosphorylate and regulate the association of GRAs such as GRA17 with the membrane (Beraki et al., 2019).

However, GRA60 is not reported to be phosphorylated in any of the available phosphoproteome analyses (Z. X. Wang et al., 2019).

Deletion of GRA60 gene in both type I and II does not impact on the progression of the lytic cycle in HFFs. Conversely, RH∆gra60 intracellular proliferation rate was slower in both IFN-γ unstimulated and stimulated MEFs compared to RH strain. The difference likely results from IFN-γ regulated effector system of the cell autonomous responses (IRGs) which are sub-

operational in human cells (Hunn et al., 2011). RH∆gra60 exhibits a significantly attenuated virulence in mice in a manner comparable to RH∆rop18 (Selleck et al., 2013). Of relevance, recent studies using CRISPR/Cas9 based screens in vivo (Young et al., 2019) and in IFN-γ stimulated macrophages (Y. Wang et al., 2019) identified GRA12 in the former study and GRA45 ,GRA46 and GRA22 as novel T. gondii virulence factors in the latter study. GRA60 was not assessed in these screens because this gene was not included in the guide RNAs libraries. GRA12, which associates with the IVN, not only contributes to parasite virulence in both type I/II strains (Young et al., 2019), but also to the establishment of chronic infection.

Corticosteroid (dexamethasone) immune suppressed or IFN-γ^-/- mice infected with parasites lacking GRA12 I/II succumb to infection. Despite its implication in modulation of IFN-γ mediated immune responses, the GTPase effectors do not assemble on the vacuoles of parasites without GRA12 (Fox et al., 2019; Wang et al., 2020). GRA7/II together with other PVM associated GRAs were found to make up the cyst wall and membrane and shown to be important for cyst formation (Guevara et al., 2020). Concordantly, homologues of T. gondii GRA60 are only found in the other cysts forming parasites, Hammondia and Neospora. GRA60 was found to decorate the cyst wall and chronically infected mice with ME49∆gra60 and ME49sag1/GRA60 displayed a notable reduction in brain cysts, which points towards a role of this protein in cyst formation. Notably, in vitro differentiated ME49pSAG1/GRA60 parasites still secreted substantial amounts of GRA60 and formed cysts, emphasizing that these conditions were not reflective of the chronic infection in the in vivo model.

Upon infection of murine cells with the avirulent ME49 and Prugniaud strains, the cytosolically abundant Irga6, Irgb10 and Irgb6 proteins (Martens et al., 2006; Martens et al., 2004) diffuse to the vacuolar membrane (Khaminets et al., 2010; Lee et al., 2020). IRGs loading at the PVM exhibits spatial hierarchy and cooperation (Hunn et al., 2008; Khaminets et al., 2010). The

pioneer Irgb6 (Khaminets et al., 2010; Lee et al., 2020), is targeted to the PVM via the universally conserved phosphate loop where it recognizes and binds to phosphatidylserine (PS) and phosphatidylinositols (PI5P) (Lee et al., 2020). Absence of Irgb6 is detrimental to loading of Irgb10, Irga6, mGBP1, mGBP2, mGBP1-5, p62 and ubiquitin onto vacuoles of avirulent T. gondii strains (Khaminets et al., 2010; Lee et al., 2020). The hierarchical sequence of docking events is proposed to be Irgb6>Irgb10>Irga6>Irgm2≅Irgd (Figure 9a) (Khaminets et al., 2010). The anti-IRGs activity in RH is attributed to the distinct yet synergistic functions of ROP5, ROP18 and GRA7 (Figure 9b) (Alaganan et al., 2014; Etheridge et al., 2014; Hermanns et al., 2016; Steinfeldt et al., 2010). ROP17 forms a complex with ROP5 and primarily phosphorylates Irgb6 independently of GRA7 (Etheridge et al., 2014) (Figure 9a and b). This study finds that GRA60/I also acts in vitro by subverting IRGs loading at the PVM. Irgb6, a key substrate of ROP5, ROP18 and ROP17, is not recruited to RH∆gra60 vacuoles (Figure 9a and b) indicating that GRA60 does not participate in the neutralization of Irgb6 via the ROP5- ROP18-ROP17 complex (Figure 9b). Contrastingly, Irgb10 and Irga6 efficiently assemble onto RH∆gra60 vacuoles regardless of Irgb6 presence. Recruitments of both Irgb10 and Irga6 on RH∆rop18∆gra60 and in RH∆rop5∆gra60 vacuoles were comparable to the single knock-out parasites indicating that GRA60, ROP18 and ROP5 work somehow in a concerted action. This is in agreement with the absence of impact of GRA60 deletion in ME49 where the inactive allele variant ROP5/II (Reese, Zeiner, Saeij, Boothroyd, & Boyle, 2011) and non-functional polymorphic ROP18/II (Saeij et al., 2006; Taylor et al., 2006) allow full recruitment of IRGs (Figure 9c). ROP5 appears to be central to the virulence complex without which the parasite resistance mechanism fails completely (Figure 9b). Irgb6, Irga6 and Irgb10 co- immunoprecipitate with ROP18 and are phosphorylated by this kinase in vitro (Fentress et al., 2010). Although ROP18 preferentially phosphorylates Irga6 (Hermanns et al., 2016; Steinfeldt

et al., 2010), it efficiently blocks Irgb6 recruitment (Fentress et al., 2010). Consequently, absence of ROP18 allows for activation and recruitment of Irgb6 and Irga6 to the PVM. In contrast, the presence of Irgb10 at the PVM of RH∆rop18 is not observed, suggesting that it might still be phosphorylated by another kinase and hence inactivated (Figure 9b). We demonstrate that GRA60 does not control the localization of ROP5 and ROP18 at the PVM or regulate the secretion and association of GRA7 with the PVM. However, upon loss of GRA60, Irgb6 is still neutralized via phosphorylation by ROP17/ROP18, whereas Irgb10 and Irga6 become activated and recruited. Previous observations showed that all Irga6-loaded vacuoles were equally positive for Irgb6 and Irgb10 (Khaminets et al., 2010) implicating the importance of both proteins for Irga6 assembly at the PVM. Our observations that Irga6 loads in presence of either Irgb6 in RH∆rop18 or Irgb10 in RH∆gra60, suggest that ROP18 and GRA60 independently hamper the Irgb6-Irga6 and the Irgb10-Irga6 pathways, respectively. In addition, RH∆rop18∆gra60 was reminiscent of RH∆rop5, making conceivable that T. gondii is using GRA60 and ROP18 in concert with ROP5 to destabilize the presence of Irga6 on the PVM.

This supposition is corroborated with the observation that GRA60 associates with ROP18 revealed by co-immunoprecipitation assay which places GRA60 as interacting partner of the virulence complex. This interaction between GRA60 and ROP18 could also be important for GRA60 association with the PVM, a similar mechanism has been observed between ROP5 and GRA7 (Hermanns et al., 2016). Both IRGs subversion tactics comparably contribute to parasite virulence in CL57/B6 mice (Alaganan et al., 2014; Selleck et al., 2013). Given the direct interaction between ROP18 and Irgb6 (Fentress et al. 2010), it is analogously plausible that GRA60 directly binds to Irgb10 in the presence of ROP5 to allow phosphorylation of Irgb10 by a yet to described kinase.

Of relevance, the extent of recruitment depends on the quantity of the IRGs available in the cytosol. The pools of IRGs are drastically reduced and inadequately docked on vacuoles in cells lacking the regulatory proteins Irgm1, Irgm2 and Irgm3 (Haldar et al., 2013; Henry et al., 2009; Hunn et al., 2008). Recruitment of Irga6 and Irgb6 to ME49 vacuoles was halved in Irgm1-/- bone marrow macrophages (Henry et al., 2009). Using the 10D7 antibody which detects GTP-bound activated Irga6, a partial reduction of Irga6 recruitment was observed on RH∆rop5 and RH∆rop18 parasites in ∆Irgm1 and ∆Irgm2 MEF cell lines compared to RH.

Contrastingly, under similar conditions, basal Irga6 loading values were observed for RH∆gra60 and ME49. It is still unclear why we observe these strain-specific differences, potentially in the context of RH∆gra60 other members of the virulence complex namely ROP5, ROP18 and GRA7, which localize normally to the PVM are still able to efficiently neutralize the available pool of Irga6 proteins. Complete negative regulation of Irga6 is observed in absence of multiple Irgm proteins (Hunn et al., 2008; Lee et al., 2020). Predictably, complete failure of Irga6 loading to the vacuole of all strains was observed in the ∆Irgm1/∆Irgm2 cell lines.

Mouse GBPs (mGBPs) exert significant anti-parasitic cell-autonomous responses against T.

gondii (Degrandi et al., 2007; Degrandi et al., 2013; Fisch et al., 2019; Haldar et al., 2013;

Kravets et al., 2016; Selleck et al., 2013; Steffens et al., 2020; Virreira Winter et al., 2011;

Yamamoto et al., 2012). Virulent T. gondii can evade mGBP1 loading via ROP5, ROP18 and ROP16 (Selleck et al., 2013; Virreira Winter et al., 2011). As alternative strategy, the secreted GRA15/II enhances parasite clearance by directly interacting with murine and human TRAF2 and TRAF6 ubiquitin ligases. This causes recruitment of IRGs, GBPs and autophagy mediated clearance in mice cells. (Mukhopadhyay et al., 2020). RH∆gra60 leads to no detectable mGBP2 recruitment while recruitment was observed in RH∆rop5 and RH∆rop18 vacuoles, suggesting that GRA60 specifically targets IRGs but not GBPs. Further studies will be

necessary to unravel the molecular mechanism by which GRA60 act as virulence factor by neutralizing the host cell autonomous defense factors Irga6 and Irgb10.

Methods

Genome mining strategy to uncover exported disordered proteins

Protein sequences of the entire T. gondii genome were extracted from ToxoDB (v42.0). Protein disorder was determined using the Linux executable version of DISpro (Cheng et al., 2005) with default settings. DISpro takes in the input protein sequence and uses a 1D-Recurrent Neural Network to predict the disorder probabilities of each residue, and classifies the disorder or order state with a threshold of probability index of 0.5. Disorder scores of the full or partial protein sequences were calculated by averaging the disorder probability scores across the sequence length. Proteins with a score >0.5 for the full-length sequence or for a sub-region of 50 aa within the first 200 aa were selected (with the exception of TGME49_273980). Here, the reasoning was that a disordered state of the protein N-terminal region might be sufficient for translocation through the PVM. The list was refined by selecting only candidate proteins displaying some degree of polymorphism between the type I, II and III lineages of T. gondii, as reflected by the ratio of substitution rates at non-synonymous and synonymous sites (dN/dS).

Relevantly, several GRA or ROP effectors contributing to parasite virulence show a dN/dS ratio > 2. Other criteria considered included the presence of a signal peptide, possession of a TM or the gene expression in the different stages of the parasite life cycle.

Bacteria, parasite and host cells maintenance

Dulbecco’s Modified Eagles Medium (DMEM, Gibco) supplemented with 5% Fetal calf serum (FCS), 25mg/ml gentamicin and 2mM glutamine was used to propagate parental and mutant strains of T. gondii tachyzoite forms in confluent human foreskin fibroblast (HFFs). We used E.coli XL-10 Gold chemo-competent bacteria to carry out all recombinant DNA experiments.

Mouse Embryonic Fibroblast (MEFs) and derived GMS knock out cell lines were maintained DMEM supplemented with 10% FCS, 25mg/ml gentamicin and 2mM glutamine.

DNA cloning and generation of constructs

Genomic DNA (gDNA) was extracted from RH∆HX∆KU80 RH and ME49∆HX∆KU80 (ME49) parasites using the Wizard SV genomic DNA purification system (Promega). GRA60 has accession number TGME49_204270 on ToxoDB, RH and ME49 gDNA was amplified using primers that span the C-terminal (Ct) of GRA60 using primers 6972 and 6973 which included KpnI and NsiI restriction sites respectively. The purified PCR product was digested with appropriate enzymes and cloned in pT8-TgMIC13-Ty-HXGPRT plasmid (Friedrich et al., 2010) between KpnI and NsiI to obtain Ct-GRA60-Ty-HXGPRT.

To produce CRISPR/Cas9 directed GRA60 knock out strains, pSAG1-CAS9-GFP- U6::sgUPRT plasmid (Shen et al., 2014) was used as PCR template to construct specific dgRNA with primers 4883/7122 and 4883/7123. Amplification was done using the Q5 site directed mutagenesis kit (NEB) according to manufacturer’s instructions. Then, a fragment of pSAG1::CAS9-GFP-U6::sgGRA60 (#7123) containing the specific sgRNA sequence was amplified using the primers 6147/6148 and subcloned into pSAG1::CAS9-GFP-U6::sgGRA60 (#7122) between the KpnI and XhoI restriction sites. KOD DNA polymerase (Novagen) was

used to amplify DHFR cassettes with primer set 7124 and 7125. These primers carry 5’ and 3’

homology sequences to GRA60, 30 base pairs long each, upstream of the gRNA sequence and after the stop codon respectively. The KOD PCR product was precipitated using sodium acetate, resuspended in 100 µl of water prior to co-transfection with the gRNA.

To produce the RHΔgra60/GRA60-Ty complemented strain, RH gDNA was amplified using primers 7135 and 6973, that span 1,5 kb of the endogenous promoter until the C-terminal (Ct) of gra60 and which included ApaI and NsiI restriction sites respectively. The purified PCR product was digested with appropriate enzymes and cloned in pT8-TgMIC13-Ty-HXGPRT plasmid (Friedrich et al., 2010) between ApaI and NsiI to obtain the Promgra60-gra60-Ty- HXGPRT vector. This vector was then linearized in the promoter region of gra60 by EcoRI enzyme.

Generation of ME49pSAG1/GRA60-Ty strain

The genomic sequence encoding the SAG1 promoter region of ME49 parasites was amplified using primer pairs 8847/8848 as described before (Di Cristina et al., 2017; Di Cristina et al., 2008). This product was sub-cloned into p-30RFP plasmid (Striepen et al., 2001) at the HpaI and BglII restriction sites to produce pSAG1-RFP-CAT. Subsequently, Myc tag epitope sequence was inserted at the tail end of sag1 promoter sequence using primers 9951/9952 using the HIFI DNA assembly approach to obtain pSAG1-Myc-RFP-CAT plasmid. Next, KOZAK sequence was added upstream of the Myc epitope tag by Q5 site directed mutagenesis to produce psag1-KOZAK-Myc-RFP-CAT. To swap the GRA60 promoter region with the promoter sequence of SAG1, KOD PCR was performed on pSAG1-Myc-RFP-CAT using primer pair 10009/100010 which contain 40 nucleotide bases mapping to the 5’ region

upstream of GRA60’s predicted promoter sequence and 3’ region encompassing sequences after the start codon. Two guide RNAs targeting the GRA60 promoter region 10013/10014 were designed and used to generate a 2-gRNA CRISPR/Cas9 plasmid as described before (Krishnan et al., 2020). 10ug SAG1 promoter cassette and 20ug of the 2gRNA plasmid were co-precipitated using sodium acetate and co-transfected into ME49. Subsequently, GFP expressing parasites were sorted by FACS analysis and cloned. The ME49pSAG1/GRA60 parasite clones were diagnosed by PCR. The integration of SAG1 promoter cassette was examined using primers 9515/10051 and 1935/10053 while loss of original GRA60 promoter region assessed using 10051/10053.

To generate the ME49pSAG1/GRA60-Ty strain, a guide RNA targeting the 3’ UTR of GRA60 was designed and inserted into plasmid by PCR using primers 4883/7123, this was co- transfected with PCR product carried out using primers 10054/10055 containing 30 base pair homology to GRA60 locus on p-GRA60-Ty. The clones were determined by IFA.

Parasite transfection and selection of stable transgenic parasites

Transfections on tachyzoite forms of T. gondii were executed by electroporation as previously described (Soldati et al., 1993). Transfectants were progressively put through selective pressure using mycophenolic acid and xanthine (MPA) for HXGPRT selection (Donald et al., 1996) or pyrimethamine (Pyr) for DHFR selection (Donald et al., 1993). All strains stably expressing the drug selection markers were cloned by limited dilution in 96-well plates. PCR and IFAs were done on single clone transgenic parasites to confirm genomic integration of constructs and expression of integrated DNA.

To generate RHGRA60-Ty strain, 40 μg of plasmid Ct-GRA60-Ty-HXGPRT was linearized by XhoI enzyme and transfected into the RH parasites. RH and ME49 Δgra60 strains were produced by co-transfecting RH, ME49, RHΔrop5 and RHΔrop18 parasite lines using 30 μg of pSAG1::CAS9-GFP-U6::dgGRA60 and 15 μg of KOD-amplified DHFR selection cassette flanked by 30 nucleotide homology regions. Single clones were obtained by limiting dilution after selection with MPA or Pyr accordingly. To generate the RHΔgra60/GRA60-Ty strain, RHΔgra60 parasites were transfected with 40 μg of the PromGRA60-GRA60-Ty-HXGPRT plasmid linearized by EcoRI enzyme. Single clones were obtained by limiting dilution after selection with MPA

Plaque assay

HFFs were seeded to form a confluent monolayer, these cells were then infected with approximately 50 freshly egressed parasites and allowed to sit for 7 days or 12 to 14 days for type I and type II strains respectively followed by fixation step using PFA/GA and plaque sizes revealed by 0.1% crystal violet (sigma).

Intracellular growth and synchronous division assay

HFFs and MEFs were infected with RH and RHΔgra60 or ME49 and ME49Δgra60 and let to grow for 24hours or 40 hours in type II infections and subsequently fixed using PFA/GA. IFAs followed using α-GAP45 antibodies. 100 vacuoles were enumerated in duplicate technical replicates, experiment was repeated three independent times, data is presented as mean ±s.d.

Assessment of synchronicity of cell division within a vacuole was carried out by labelling parasites using α-IMC1 antibodies. The state of daughter cells division was captured by fixation at 30 hours post invasion for type I RHΔKU80 and RHΔgra60 and 40 hours post invasion for type II ME49 and ME49Δgra60 strains. 100 vacuoles were enumerated per experiment, experiments were repeated 3 independent times and results presented as mean ±s.d.

Antibodies

Antibodies listed below were used in either both IFA or Western blot analysis, their sources and previous methods of employment are described herein; polyclonal rabbit α-catalase, α- IMC1 and α-GAP45, mouse monoclonal α-Ty (BB2) (Frenal et al., 2014). Mouse monoclonal α -ROP5, α -ROP2,3,4, α -ROP1, α-GRA1 and α-GRA3 were generously donated by Dr J.-F.

Dubremetz. α -HA, α -Irga6, α-Irgb6, α-Irgb10 and α-Irgd antibodies were kind gifts from Prof.

J.C. Howard and Dr. Tobias Steinfeldt (Steinfeldt et al., 2010). Rabbit polyclonal α -ROP 18 antibodies were produced by Eurogentec. Rabbit polyclonal α -Ty, was donated by Tonkin C.J.

Polyclonal rabbit antibodies α -mgb2 catalog number 11854-1-AP and α -hGB1 catalog number 15303-1-AP were purchased from (Proteintech). Secondary peroxidase conjugate goat α-rabbit/mouse antibodies (Molecular probes) were used for Western blot analyses.

Secondary antibodies Alexa Fluor 488- and Alexa Fluor 594-conjugated goat α-mouse/rabbit antibodies (Molecular probes) were used for IFAs.

Immunofluorescence Assay

HFFs were seeded on coverslips and allowed to form confluent monolayer. These were then infected with appropriate strains and fixed with 4% paraformaldehyde (PFA)/0.05%

glutaraldehyde (GA) for 10 min or 20 mins for GRAs PV and PVM localization followed by a quenching step in 0.1M glycine/PBS. Infected cells were then permeabilized with 0.2% Triton X-100/PBS (PBS/Triton) succeeded by a blocking step in with 3% BSA in PBS. An incubation step for 1 hour with primary antibodies diluted in 1% BSA/PBS was done followed by (3x5 minutes) PBS washes. Next, coverslips were incubated for 1hour with the secondary antibodies described above diluted in 1%BSA/PBS solution. Parasite and host cell nuclei were then stained by incubation in DAPI (4’,6-diamidino-2-phenylindole; 50μg/ml in PBS) for 7 minutes. Final (3x5 minutes) PBS washes preceded mounting of coverslips on slides using Fluoromount G (Southern Biotech) and slides stored at 4°C in the dark. Confocal images were taken using Zeiss microscopes (LSM700 or LSM800 Airyscan objective apochromat 63x /1.4 oil) found at the Bioimaging core facility of the Faculty of Medicine, University of Geneva. Z- stack sections were processed using the ImageJ software.

ROP5 and ROP18 localization at PVM

10 minutes post invasion, vacuoles were either permeabilized using 0.01% saponin for ten minutes. This was followed by IFA using GRA3 antibodies as a marker for the PV of an invaded parasite and either ROP5 or ROP18 antibodies.

GRA60 kinetics at the PVM

RHGRA60-Ty parasite vacuoles were permeabilized using 0.01% saponin for ten minutes at 5 ,10, 30 and 60 minutes post invasion. This was followed by IFA using ROP1 antibodies as a marker for the PVM of an invaded parasite and either Ty antibodies.

GRA60 topology at PVM

RHGRA60-Ty parasite vacuoles were either selectively or fully permeabilized using 0.001%

saponin for five minutes or with 0.2% Triton X-100 for 10 minutes respectively. This was followed by IFA using GAP45, ROP2,3,4 and Ty antibodies.

Co-immunoprecipitation assay

HFFs were infected with RHGRA60-Ty tachyzoites for 20 hours. The infected cells were washed with phosphate-buffered saline (PBS), scraped off and lysed in 1.5 ml of lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris/HCl (pH 8), 5 mM EDTA supplemented with protease inhibitors for 2 h under constant rotation at 4 °C. GRA60 was immunoprecipitated by incubating lysates with Ty antibodies at 4 °C followed by an additional 1 h of incubation with 100 µl 1:1 (lysis buffer) bead suspension of protein A-Sepharose (Amersham) resin. Beads were washed three times with lysis buffer, boiled in for 5 min at 95 °C in SDS loading buffer with DTT and then subjected to western blot. The efficiency of GRA60 precipitation was investigated by probing with mouse anti-Ty antibodies and detected by western blot as described in the methods section.

Western blot analyses

For fractionation, pelleted intracellular and extracellular parasites were resuspended in the following solutions containing protease inhibitor; PBS, 0.1M Sodium carbonate in PBS, 1%

Triton X-100 in PBS or RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1%

SDS, 50 mM Tris pH 7.5) incubated on ice for 15 minutes and then centrifuged at 14,000 r.p.m.

at 4 °C. The supernatant from each of the preparations above was collected in a separate tube and named (S), the (S) tubes and remaining membrane containing pellet fractions (P) were mixed with SDS–PAGE loading buffer under reducing conditions. The proteins in the (S) and (P) portions were transferred to nitrocellulose membranes and probed with appropriate antibodies in 5% non-fat milk powder dissolved in PBS-0.05% Tween20. Bound secondary peroxidase-conjugated antibodies were revealed using the ECL systems (Amersham).

Competition assay

Wildtype and Δgra60 parasites of both type I and II lineages were mixed with GFP-expressing parasites in an estimated ratio of about 80:20 respectively. HFFs were infected with these preparations and parasite proportions progressively measured over 5 passages by Flow cytometry. Briefly, at each passage 200µl of freshly egressed parasites were collected in an eppendorf tube and incubated with Hoechst DNA stain in 1/1000 dilution. A fixation step with 200µl of with 4% paraformaldehyde (PFA)/0.05% glutaraldehyde (GA) for 10 min ensued. Next the parasites were centrifuged at 2000 rpm for 5 minutes, the fixative removed and resuspended in 500µl 0.1M glycine/PBS solution. The cells were gated bases on DNA labelling and GFP expression using the Analyser 3 Laser Gallios 4 instrument and anaysis done using the Kaluza software provided at the Flow Cytometry Facility Platform at University of Geneva.

Data is presented as mean±s.d of 3 independent experiments.

IRG and mGBP2 recruitment assay

Mouse Embryonic Fibroblast (MEFs), MEF∆Irgm1, MEF∆Irgm2 and MEF∆Irgm1∆Irgm2 were generous donations from Prof. Masahiro Yamamoto. The host cells were seeded on coverslips and allowed to form confluent monolayer. 1000U/ml of mouse IFN-γ catalogue number GFM16-20 purchased from Cell Guidance Systems (CGS) was added to the cells for approximately 20hours. 250µl volume of 5x10⁶ parasites/ml freshly egressed tachyzoites was used to infect the IFN-γ activated cells. The infected coverslips were centrifuged at 1100rpm for 30 seconds and parasite allowed to invade for 20 minutes at 37°C. The non-invaded parasites were washed off using DMEM, fresh medium added and left to incubate at 37°C for 1 hour. The cells were subsequently fixed with 4% PFA in PBS for 20 minutes, PFA was removed and replaced with PBS. Next, the cells were semi-permeabilized using 0.02%

saponin in PBS for 10 minutes, blocked using 3%BSA in PBS for 20 minutes. IFA was carried out using the primary IRGs and mGBP2 antibodies described in section above diluted in 1/1000 in 1%FCS in PBS solution. GRA1 positive vacuoles were assessed for co-labelling with murine IRGs or GBP antibodies and proportions enumerated in 100 vacuoles in duplicates per experiment. The experiments were repeated independently at least 4 times and data presented as mean±s.d. Images were taken and processed as described in IFA section.

Acute virulence

Six weeks old female C57BL/6 mice provided by Charles River Laboratories were infected intraperitoneally with tachyzoite forms. Five mice per test group were used for the virulence experiments. The progression of infection was monitored daily and appearance symptoms

characteristic of acute toxoplasmosis (bristled hair, inability to eat or drink and complete prostration) used as cues to sacrifice animals upon presentation.

Cyst counting from processed brain tissue

8 C57BL/6 mice per test group were infected with 250 tachyzoites and surviving mice were sacrificed 5 weeks post infection. Brains of mice were smashed in 1 ml of 1% Tween in PBS by sequential passing 5X through a 18G needle, 10X through a 20G needle and finally 10X through the 23G to produce a homogeneous solution. Cysts were enumerated from 5 portions of 10µl volumes per sample using the 20X objective lens of an inverted microscope.

Ethics statement

All experiments were carried out under license number governed by the Swiss Federal Veterinary Office rules and regulations.

Statistical analysis

GraphPad Prism software was used to perform statistical analysis. The data presented are the means and ±s.d for at least three independent experiments. Significance of differences in means between groups was compared using the Student’s paired t test. The threshold used to set the cut off for statistical significance was Ps < 0.05.

Acknowledgements

This work was supported by the Swiss National Foundation Sinergia CRSII3_160702 and by European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant agreement no. 695596. This work was also supported by the CARIGEST SA grant awarded to DS-F. We thank Tobias Steinfeldt of the Faculty of Medicine, University of Freiburg for support and advice. We are also grateful to Tobias Steinfeldt and Jonathan C. Howard for providing us with the IRG antibodies and methods used in this study.

We are grateful to Matteo Lunghi for helping with the animal experiments, Dr. Gaelle Lentini’s advice and analytical support and to Dr. Aarti Krishnan for critical reading of the manuscript.

Author contributions

MN, PHM, SY,JM and JBM conducted the experiments. DSF supervised and coordinated the project. MN, PHM and SY analyzed the data. MN, PHM, SY, MY and DSF conceived and designed experiments. MN, PHM, SY, MY and DSF wrote the manuscript.

Data Availability statement

The data that supports the findings of this study are available in the supplementary material of this article. The data that support the findings of this study are available from the

corresponding author upon reasonable request.

Conflict of interest

All authors listed declare no conflict of interest

Figure Legends

Figure 1 GRA60 is an intrinsically disordered protein secreted to the PV and PVM of T.

gondii

(a) Schematic representation of the full length GRA60 protein (544 amino acids). Signal peptide (SignalP) appears in yellow, the 3 intrinsically disordered regions (DISPRO 1.0) IDR1, IDR2 and IDR3 in grey and the transmembrane domain (TMHMM) in blue. Values above 0.5 are predicted to be disordered and values below 0.5 correspond to folded domains. (b) RHGRA60-Ty extracellular parasites were embedded on coverslips using polylysine, fixed and IFAs carried out using α-GRA1 and α-Ty antibodies localizing GRA60 to dense granules. HFFs infected with RHGRA60-Ty for 24 hours were fixed and IFAs carried out using α-Ty, α-GAP45 and α-GRA3 antibodies show GRA60 is secreted to the PV space and PVM in intracellular parasites. Scale bar = 5μm. (c) Preparations of HFFs extractions co-infected with RHGRA60- Ty and RHCDC50.1mAID.3HA for 24 hours and freshly egressed extracellular parasites, were harvested, mechanically lysed and membrane enriched fractionation treated with PBS, 1%

Triton X-100, 0.1 M sodium carbonate or RIPA buffer solutions containing protease inhibitors, followed by a centrifugation step to obtain insoluble pellet (P) or soluble portion (S). Western blot was carried out on the fractions as indicated with α-catalase antibody as a control for soluble proteins. (d and e) HFFs infected with RHGRA60-Ty for 24 hours were fixed and

permeabilized with 0.001% saponin (d) and 0.2% Triton X-100 (e). IFAs carried out using α- Ty, α-GAP45 and α-ROP2,3,4 antibodies show C-terminus of GRA60-Ty faces the PV lumen parasites. Scale bar = 5μm. (f) Schematic representation of GRA60 localization within the parasite, in the vacuolar space and topology on the PVM.

Figure 2 GRA60 contributes to parasite fitness in mouse but not human cells in vitro

(a) Deletion of GRA60 has no impact on the lytic cycle in type I and II strains, determined by equally sized plaques formed 7 and 12 days post infection on confluent HFFs. (b) Assessment of intracellular growth at 24 hours and 40 hours post infection in HFFs with parasites lacking GRA60 in both type I and II strains show non aberrant replication rates. (c) Lack of impaired fitness in vitro confirmed by competition assay using GFP-expressing parasites as an internal control for both RH and ME49 mutants over 5 and 4 successive lytic cycles respectively. (d) IFAs using IMC1 antibodies to stain mother and daughter cells 30 and 44 hours post infection show asynchronous division in RHΔgra60 and ME49Δgra60 parasites. Scale bar = 5μm (e) Quantification of synchronously dividing parasites in both type I and II Δgra60 parasites 30 and 44 hours p.i. Results are means ± s.d of 3 independent experiments. Unpaired statistical significance test applied *p<0.05. p values in (Table S3). (f) Assessment of intracellular growth at 24 hours post infection in MEFs activated or not with IFN-γ (1000U/ml) for 20 hours. MEFs were infected with RH or RHΔgra60 for 24 hours and IFA performed using α-GAP45 antibodies. The number of parasites per vacuole was enumerated and is shown as percentage.

Mean ±s.d, n= 4 experiments.

Figure 3 GRA60 contributes to parasite virulence during acute infection and chronic stages in mice

(a) 200 tachyzoites of RH and RHΔgra60 strain and (b) 1000 of ME49 and ME49Δgra60 were intraperitoneally injected in C57BL/6 mice (n=5) and mice survival was subsequently monitored over time. Mice infected with RHΔgra60 show a median time to death of 17 days compared to 9 for RH group. The difference between survival curves was statistically significant for the type I strains (p= 0.0255), but not for type II strains was not (p=0.1266) (Table S3), p values from the Gehan-Breslow-Wilcoxon test. (c) The cyst burden was evaluated at 5 weeks following IP injection of C57BL/6 mice (n=8) with 250 ME49 and ME49Δgra60 tachyzoites, median ME49 =1533, n=7, median ME49Δgra60 = 580, n=5, p= 0.202 (Table S3).

(d) Similarly, the cyst burden was assessed in the brain of ME49pSAG1/GRA60 infected mice, median ME49 = 3636, n=4, median ME49pSAG1/GRA60 = 850, n=6, p= 0.0381 (Table S3).

Figure 4 GRA60 is involved in neutralization of Irgb10 and Irga6 but not Irgb6 at the PVM

The Irg proteins recruitment was evaluated on MEFs stimulated with IFN-γ (1000U/ml) for 20 hours prior to infection for 90 minutes with indicated strains.

Representative examples of Irgb10 (a), Irga6 (c) and Irgb6 (e) recruitment at the PVM of RHΔrop18 and RHΔgra60 vacuoles stained with GRA1 antibodies. Scale bar =10μm.

Quantification of Irgb10 (b), Irga6 (d) and Irgb6 (f) positive vacuoles per indicated strain. Mean

±s.d, n = 3 experiments Irgb10, (Irga6 and Irgb6) n = 4 experiments. Unpaired statistical significance test was applied, p values available in Table S3.

Figure 5 GRA60 display fast secretion kinetics and does not alter localization of ROP5, ROP18 and GRA7 on the PVM

(a, c and e) The localization of ROP5 (a), ROP18 (c) and GRA7 (e) on the PVM at 10 minutes post invasion with RH and RHΔgra60 parasites was observed by IFA. Infected HFFs were permeabilized with 0.01% saponin and PVM was stained using the GRA3 marker. (b, d, and f) Quantification of mean fluorescence intensity of ROP5 (b), ROP18 (d) and GRA7 (f) on vacuoles of RH and RHΔgra60 parasites. Unpaired student’s t test was applied and p values are available in Table S3). (g) Secretion kinetics of GRA60-Ty in infected HFFs fixed at 5, 10, 30 and 60 minutes post invasion. Vacuoles were permeabilized using 0.01% saponin. ROP1 was used as a marker of an early discharged rhoptry protein. Scale bar = 5μm. Mander’s Colocalization Coefficients of ROP1 and GRA60-Ty are available in Table S3.

Figure 6 GRA60 interacts with ROP18, a member of the ROP5/ROP17/GRA7 complex

(a) RHGRA60-Ty was immunoprecipitated with mouse anti-Ty antibodies in infected HFFs.

Supernatant (SN), pellet (P) and immunoprecipitate (IP). Immunoblot presents efficiency of GRA60 immunoprecipitation using Ty antibodies. (b) RH and RHΔrop18 parasite lysates were separated on SDS-PAGE and ROP18 migration profile was analyzed by western blot. ROP18 migrates at the size of 55 kDa and Actin was used as a loading control (c) Immunoblots probing the interaction of GRA60 with ROP18 following co-immunoprecipitation of RHGRA60-Ty in infected HFFs. Supernatant (SN), pellet (P) and immunoprecipitate (IP). ROP18 is detected at expected size and becomes enriched in the IP. Catalase was used as a loading control.

Immunoblot representation of three similar experiments.

Figure 7 Loss of Irga6 recruitment on RHΔgra60 PVM in MEFS without Irgm1 and Irgm2 proteins

(a) Schematic representation of Irgm1 and Irgm2 proteins regulation of Irga6 in murine cells in absence or presence of avirulent T. gondii parasites (b) Quantification of Irga6 positive vacuoles in ME49, RH, RHΔrop5, RHΔrop18 and RHΔgra60 parasites in MEFs (MEFSV40, ΔIrgm1, ΔIrgm2 and ΔIrgm1.ΔIrgm2) stimulated with IFN-γ 1000U/ml for 20 hours. The counting was done at 90 minutes post infection. Mean ±s.d, n= 3 experiments. Unpaired statistical significance test applied *p<0.05. p values are available in Table S3.

Figure 8 GRA60 is not involved in subversion of mGBP2 recruitment on the PVM

(a) Localization of mGBP2 on the PVM in infected MEFs observed by IFA. MEFs were stimulated with 1000U/ml IFN-γ for 20 hours and infected with ME49, RH, RHΔrop5, RHΔrop18 and RHΔgra60 parasites for 90 minutes. The vacuole was stained using GRA1 as marker.

Scale bar = 10μm. (b) Quantification of mGB2 positive vacuoles per indicated strain in IFN-γ activated MEFs at 1000U/ml concentration for 20 hours. The counting was done at 90 minutes post infection. Mean ±s.d, n= 3. Unpaired statistical significance test applied *p<0.05. p values are available in Table S3.

Figure 9 Model for IRG proteins recruitment to virulent, avirulent and attenuated virulence strains of T. gondii

(a) IRG proteins loading observed on vacuoles of listed T. gondii strains as observed in this and other studies (Alaganan et al., 2014; Etheridge et al., 2014; Hermanns et al., 2016). The docking process at the PVM is hierarchical and cooperative, initiated by Irgb6 followed by Irgb10 then Irga6 (Khaminets et al., 2010; Lee et al., 2020). (b) Binding and phosphorylation (P) of IRG proteins by virulence complex members ROP5, ROP18, GRA7 and ROP17 disturb IRG mediated parasite clearance (Alaganan et al., 2014; Etheridge et al., 2014; Hermanns et al., 2016; Steinfeldt et al., 2010). In RH∆rop5, Irgb6, Irgb10 and Irga6 efficiently load onto PVMs. In RH∆rop18, the ability to block Irgb6 and Irga6 is lost but can efficiently prevent Irgb10 from loading onto the vacuoles. In RH∆gra60, Irgb6 is avoided but Irgb10 and Irga6 still load onto vacuoles regardless. (c) Recruitments events in ME49 strain comparable to RH∆rop5. In ME49 strain allelic variations in ROP5 (Reese et al., 2011) and polymorphisms ROP18 (Saeij et al., 2006; Taylor et al., 2006) render these proteins inactive (lighter shade). In ME49∆gra60, no differences are observed from the parental line.

Supplementary figures

Table S1 Features of candidate proteins

Accession numbers, protein sequences, dNdS ratio (Non-synonymous/Synonymous SNPs), CRISPR score and transcriptomics RNA-Seq data were obtained from ToxoDB (toxodb.org).

For the transcriptomics RNA-Seq data, negative values indicate a lower expression in given stage compared to tachyzoites and positive values indicate a higher expression in a given stage compared to tachyzoites. Disorder prediction was obtained with DISpro 1.0 software.

Values above 0.5 are predicted to be disordered and values below 0.5 correspond to folded domains. The score was calculated for the full-length protein as well as for the 200 first aa, divided in 4 groups of 50 aa. Molecular weight in kilo Dalton (kDa). Actin (ACT1) is included in the list as an example of a protein with very low probability of being disordered.